Neutrophil chemotactic factor cloned cDNA and monoclonal antibodies thereto

ABSTRACT

An isolated, synthetic preparation of a novel neutrophil-specific chemotactic factor (NCF), monoclonal antibodies having specific binding affinity for NCF and a clone containing the complete cDNA coding sequence for NCF are disclosed.

This application is a division of Ser. No. 08/818,631 filed Mar. 14, 1997, which is a division of Ser. No. 07/169,033 filed Mar. 16, 1988 now U.S. Pat. No. 5,652,338.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to an isolated, synthetic preparation of a novel neutrophil-specific chemotactic factor (NCF), monoclonal antibodies having specific binding affinity for NCF and a clone containing the complete coding sequence for NCF.

2. State of the Art

Activated monocytes/macrophages produce various mediators that cause inflammation. Among them are chemotactic factors which cause white blood cells to migrate into inflammatory sites where these factors are released. Neutrophils, the dominant leukocytes attracted by the chemotactic factors, are believed to play a critical role in the inflammatory reactions. Such diseases as rheumatoid arthritis, idiopathic pulmonary fibrosis and certain pathological inflammatory changes in many other conditions are believed to be caused by neutrophils and/or their products. However, a specific pro-inflammatory mediator released by tissue macrophages and other cells in response to inflammatory stimuli and leading to neutrophil-rich leukocyte accumulation in host defense and disease, has not heretofore been identified and isolated.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to provide a biologically active novel synthetic polypeptide acting as a neutrophil-specific chemotactic factor (NCF).

It is a further object of the present invention to provide a molecular clone containing the complete coding sequence for the synthesis of NCF by either prokaryotic or eukaryotic expression vectors.

It is a still further object of the present invention to provide monoclonal antibodies having specific binding affinity for NCF of the present invention.

It is another object of the present invention to provide a kit comprising a container containing the cDNA for NCF quantitation, detection or localization of NCF mRNA in a body sample.

It is yet another object of the present invention to provide a kit comprising a container containing anti-NCF antibodies having specific binding affinity for NCF for quantitation, detection or localization of NCF in a body sample.

Other objects and advantages will become evident from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, features and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawings wherein:

FIG. 1 demonstrates translation of cDNA into NCF protein in reticulocyte lysate system; and

FIG. 2 shows:

(a) Northern blot analysis of mRNA induction in lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cells (PBMC);

(b) The time course of the accumulation of neutrophil chemotactic activity in culture media of PBMC after stimulation with LPS;

(c) Induction of NCF mRNA in PBMC by IL 1 or TNF, but not by IL 2, gamma-IFN, and alpha-IFN;

(d) HPLC gel filtration analysis of IL 1 and TNF induced neutrophil chemotactic activity.

DETAILED DESCRIPTION OF THE INVENTION

The above and various other objects and advantages of the present invention are achieved by a homogeneously pure, isolated, synthetic neutrophil chemotactic protein, designated herein NCF, composed in the whole or in part only of the following amino acid sequence (single letter code):

NH₂-S-A-K-E-L-R-C-Q-C-I-K-T-Y-S-K-P-F-H-P-K-F-I-K-E-L-R-V-I-E-S-G-P-H-C-A-N-T-E-I-I-V-K-L-S-D-G-R-E-L-C-L-D-P-K-E-N-W-V-Q-R-V-V-K-F-L-K-R-A-E-N-S (SEQ ID NO:2)

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed herein are standard methodologies well known to one of ordinary skill in the art.

Chemical synthesis of the NCF of the present invention composed of the 72 amino acid residues as shown above, is achieved by commercially available polypeptide synthesizers. Alternatively, the NCF of the present invention is synthesized by standard techniques employing an expression vector containing in its genome the cloned complete coding sequence of NCF. Anti-NCF monoclonal antibodies of the present invention are prepared by standard hybridoma technology and utilized for purification and assaying purposes following standard immunological methodologies well known in the art.

High performance liquid chromatography (HPLC), in situ hybridization assays, Northern blotting analyses and the like are typical examples of the standard conventional techniques well known to one of ordinary skill in the art, which can be employed for isolation, localization, differentiation, detection, or measurement of the mRNA for NCF in biological samples.

It should be noted that the fact that chemically synthesized polypeptide of the present invention at 10 nanomolar concentrations acts as a neutrophil attracting factor, is shown by the results in Table 1.

TABLE 1 Chemotactic response of human neutrophils to chemically synthesized NCF. Percentage of assay neutrophils Concentration of NCF, nanomolar that migrated 1000 23 100 34 10 32 1 5 0.1 1 Hanks medium 0.3 10⁻⁷M fMet-Leu-Phe 40 1. It is typical for chemoattractant dose-response curves to show an optimum, with a decreased response at concentrations above the optimum. 2. fMet-Leu-Phe is a commonly used reference chemoattractant.

It may be pointed out that various stimuli cause the release or secretion of more than one chemoattractant. Without the cDNA of the present invention, it is clear, of course, that the presence, specifically of the mRNA for NCF as an involved factor in a particular clinicopathological situation, could not be definitively identified and diagnosed. cDNA of the present invention due to its binding affinity for mRNA for NCF, for the first time makes it possible to analyze body samples such as joint fluid, sputum, alveolar lavage fluid, tissue samples and the like to detect the presence or absence of mRNA for NCF. Of course, the antibodies can also be utilized for diagnostic purposes to detect the NCF and to neutralize the NCF for alleviating any disease or anomalous conditions in which the presence of NCF is found to be a causative factor.

A pharmaceutical composition for use in treating inflammatory condition comprises an anti-inflammatory effective amount of the anti-NCF monoclonal antibodies in pharmaceutically acceptable carrier, such as physiological saline, sterile non-toxic buffer and the like.

A deposit of cDNA for NCF and of the hybridoma for anti-NCF monoclonal antibodies have been made at the ATCC, 10801 University Boulevard, Manassas, Va. 20110-209 on Jan. 12, 1988 and Feb. 17, 1988, respectively, under the accession numbers 40412 and HB9647, respectively. The deposits shall be viably maintained, replacing if they became non-viable, for a period of 30 years from the date of the deposit, or for 5 years from the last date of request for a sample of the deposit, which is longer, and made available to the public without restriction in accordance with the provisions of the law. The Commissioner for Patents and Trademarks, upon request, shall have access to the deposits.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

2 1 1560 DNA homo sapiens 1 ctccataagg cacaaacttt cagagacagc agagcacaca agcttctagg acaagagcca 60 ggaagaaacc accggaagga accatctcac tgtgtgtaaa catgacttcc aagctggccg 120 tggctctctt ggcagccttc ctgatttctg cagctctgtg tgaaggtgca gttttgccaa 180 ggagtgctaa agaacttaga tgtcagtgca taaagacata ctccaaacct ttccacccca 240 aatttatcaa agaactgaga gtgattgaga gtggaccaca ctgcgccaac acagaaatta 300 ttgtaaagct ttctgatgga agagagctct gtctggaccc caaggaaaac tgggtgcaga 360 gggttgtgga gaagtttttg aagagggctg agaattcata aaaaaattca ttctctgtgg 420 tatccaagaa tcagtgaaga tgccagtgaa acttcaagca aatctacttc aacacttcat 480 gtattgtgtg ggtctgttgt agggttgcca gatgcaatac aagattcctg gttaaatttg 540 aatttcagta aacaatgaat agtttttcat tgtaccatga aatatccaga acatacttat 600 atgtaaagta ttatttattt gaatctacaa aaaacaacaa ataattttta aatataagga 660 ttttcctaga tattgcacgg gagaatatac aaatagcaaa attgggccaa gggccaagag 720 aatatccgaa ctttaatttc aggaattgaa tgggtttgct agaatgtgat atttgaagca 780 tcacataaaa atgatgggac aataaatttt gccataaagt caaatttagc tggaaatcct 840 ggattttttt ctgttaaatc tggcaaccct agtctgctag ccaggatcca caagtccttg 900 ttccactgtg ccttggtttc tcctttattt ctaagtggaa aaagtattag ccaccatctt 960 acctcacagt gatgttgtga ggacatgtgg aagcacttta agttttttca tcataacata 1020 aattattttc aagtgtaact tattaaccta tttattattt atgtatttat ttaagcatca 1080 aatatttgtg caagaatttg gaaaaataga agatgaatca ttgattgaat agttataaag 1140 atgttatagt aaatttattt tattttagat attaaatgat gttttattag ataaatttca 1200 atcagggttt ttagattaaa caaacaaaca attgggtacc cagttaaatt ttcatttcag 1260 atatacaaca aataattttt tagtataagt acattattgt ttatctgaaa ttttaattga 1320 actaacaatc ctagtttgat actcccagtc ttgtcattgc cagctgtgtt ggtagtgctg 1380 tgttgaatta cggaataatg agttagaact attaaaacag ccaaaactcc acagtcaata 1440 ttagtaattt cttgctggtt gaaacttgtt tattatgtac aaatagattc ttataatatt 1500 atttaaatga ctgcattttt aaatacaagg ctttatattt ttaactttaa aaaaaaccgg 1560 2 72 PRT homo sapiens 2 Ser Ala Lys Glu Leu Arg Cys Gln Cys Ile Lys Thr Tyr Ser Lys Pro 1 5 10 15 Phe His Pro Lys Phe Ile Lys Glu Leu Arg Val Ile Glu Ser Gly Pro 20 25 30 His Cys Ala Asn Thr Glu Ile Ile Val Lys Leu Ser Asp Gly Arg Glu 35 40 45 Leu Cys Leu Asp Pro Lys Glu Asn Trp Val Gln Arg Val Val Glu Lys 50 55 60 Phe Leu Lys Arg Ala Glu Asn Ser 65 70 

What is claimed is:
 1. A kit for determining the level of NCF mRNA-present in a sample, comprising a container containing cDNA for NCF for performing assay to determine the level of mRNA for NCF in a biological sample.
 2. A method for detecting the level of mRNA for NCF in a sample, comprising reacting said sample with cDNA and determining the occurrence of hybridization by any conventional technique, the presence of said cDNA-mRNA hybrids being indicative of the presence of mRNA for NCF in said sample. 